Pharmaceutical composition for preventing or treating osteoporosis or obesity comprising phenyltetrazole derivative

ABSTRACT

A phamaceutical composition comprising a phenyltetrazole derivative of formula (I) or a pharmaceutical acceptable salt thereof is effective in preventing or treating osteoporosis, obesity, diabetes, or hyperlipidemia, by regulating protein TAZ. 
     
       
         
         
             
             
         
       
         
         
           
             wherein, A is ethyl or n-butyl; R 1  is methyl, —CH 2 OH, —CO 2 CH 3 , —CH 2 F, —CH(OCH 3 ) 2 , —CH 2 OC(═O)CH 3 , styryl or —CH 2 OCH 2 SCH 3 ; R 2  is H, Br, —CO 2 CH 3 , phenyl, pyridin-2-yl, pyridin-3-yl, 
           
         
       
    
     
       
         
         
             
             
         
       
         
         
           
              or styryl; R 3  is H or methyl; X is CH or N; and P is H or —CH(CH 3 )OCH 2 CH 3 .

FIELD OF THE INVENTION

The present invention relates to a pharmaceutical composition forpreventing or treating osteoporosis, obesity, diabetes, orhyperlipidemia, comprising a phenyltetrazole derivative or apharmaceutical acceptable salt thereof as an active ingredient.

BACKGROUND OF THE INVENTION

Senile diseases such as osteoporosis are becoming preponderant with anever increasing number of the elderly people. Osteoporosis is a bonedisease that causes reduced bone mineral density (BMD), disrupted bonemicroarchitecture, and alteration of the amounts various proteins inbone, leading to an increased risk of fracture. Osteoporosis is causedby an imbalance between bone resorption and bone formation andprogresses when bone resorption outpaces bone formation. Withosteoporosis, the calcified bone tissue density decreases, causing theexpansion of marrow cavity. As the symptoms progress, bone fractureoccurs easily even by a slight stressful impact.

Early studies for osteoporosis had focused mainly on calcium andphosphorus metabolic disorders, but failed to define the pathogenesisthereof. The existing therapeutic agents for treating osteoporosis arerepresented by bisphosphonate products (e.g., Alendronate andEtidronate), hormone products (e.g., raloxifene), vitamin D products,calcitonin products, and calcium products. However, bisphosphonateproducts have the problems of a low absorption rate, a complicatedmethod of administration, and the tendency to induce esophagitis.Hormone products require life-time administration, which may cause sideeffects such as breast cancer, uterine cancer, cholelithiasis, andthrombosis. Vitamin D products are expensive but not much effective.Calcitonin products have also the problems of high costs and an uneasymethod of administration. Calcium products have fewer side effects butare limited to prevention rather than treatment. Short-termadministration of a drug is not much effective for the treatment ofosteoporosis, requiring long-term administration of a drug. Therefore, anovel drug causing much reduced side effects and having enhancedmedicinal effects is required for long-term administration.

Bone marrow-derived adult stem cells, particularly mesenchymal stemcells (MSCs), are of a pluripotent cell type that differentiates intoseveral distinct cell lineages, such as osteocytes, chondrocytes,myocytes, and adipocytes. The differentiation of adult stem cells tendsto reduce the age-related diseases, to prevent: the reduction in theregeneration of bone, cartilage, and muscle tissues; the decline of theimmune function; and environment-caused diseases. Thus, a method foractivating the differentiation of adult stem cells may be an efficacioustherapy for diseases associated with metabolic diseases, bone diseases,and aging.

The differentiation of adult stem cells into a specific cell type iscontrolled by an important transcription factor, the expression of whichis regulated by the interaction with the outer signal transduction. Inparticular, adipocyte differentiation is known to be regulated bytranscription factor PPARγ (peroxisome proliferation-activated receptorγ). The transcription factor facilitates or inhibits the adipocytedifferentiation by binding with different ligands, and an increase inthe activity of the transcription factor is known to facilitate theadipocyte differentiation, leading to increased obesity [MacDougald etal., Annu. Rev. Biochem., 1995; 64:345-73; Adams et al., J. Clin.Invest., 1997; 100:3149-53; Fajas et al., Curr. Opin. Cell Biol., 1998;10:165-73].

Recently, a transcription coactivator, protein TAZ (transcriptioncoactivator with PDZ-binding motif), which regulates the transcriptionfactor PPAR γ has been identified [Kanai et al., Embo. J., 2000;19:6778-91]. The TAZ protein is cloned as a partner protein which bindsto 14-3-3 proteins, and it is phosphorylated at serine 89, whichinteract with 14-3-3 proteins in the cytosol [Kanai et al., Embo. J.,2000; 19:6778-91; Park et al., J. Biol. Chem., 2004; 279:17384-90]. TheTAZ protein contains WW domains, coiled-coil domains, and PDZ-bindingmotifs, which suggests the possibility of various binding with otherproteins. In particular, the WW domains show strong binding affinitywith peptide sequence PPXY, which suggests the possibility of the TAZprotein to bind with several proteins containing PPXY motifs. In 2003,it was found that the WW domains in protein TAZ bind with RUNX2(runt-related transcription factor 2) which is a decisive regulatoryfactor to facilitate the osteoclast differentiation. It was reportedthat the expression regulatory activity of RUNX2 target gene isamplified and the expression of bone-specific gene increases, leading toan osteogenic facilitation, through the binding [Hong et al., Science,2005; 309:1074-8]. Further, polymavirus T antigen, a protein binding tothe WW domains in protein TAZ is known, but its precise function incells is not clearly understood. Furthermore, PPARγ, one oftranscription factor having PPXY motif, has been identified as a novelTAZ binding protein, and such binding inhibits the adipocytedifferentiation by PPARγ [Hong et al., Science, 2005; 309:1074-8]. Themechanism of protein TAZ for the inhibition of adipocyte differentiationis explained by the fact that protein TAZ binds with PPARγ not only toinhibit the DNA binding activity of PPARγ but also to inhibit the genetranscription facilitation activity, thus inhibiting the expression ofadipocyte-specific PPARγ target gene. The binding of protein TAZ withRUNX2 and PPARγ plays a significant important role in regulating thedifferentiation of MSCs. Specifically, it has been found in thedifferentiation of MSCs that osteoclasts differentiation is facilitated,while the adipocyte differentiation is regressed by the binding ofprotein TAZ with proteins RUNX2 and PPARγ, [Hong et al., Science, 2005;309:1074-8; Deng et al., Front Biosci., 2008; 13:2001-21; Hong et al.,Cell Cycle 2006; 5:176-179]. That is, the differentiation of protein TAZdetermines the differentiation of MSCs.

TBX5 (T-box transcription factor 5) is known as another TAZ bindingprotein, and the binding thereof is understood to play an important rolein the limb and cardiac formation [Murakami et al., Proc. Natl. Acad.Sci., USA., 2005; 102:18034-9]. Further, TBX5 possibly binds withprotein PAX3 important in the early embryonic phase to regulate itsfunction [Murakami et al., Biochemical & Biophysical ResearchCommunications, 2006; 339:533-9]. In addition, protein TAZ shows itsactivity by binding with various PDZ domain-containing proteins throughPDZ-binding motif. TTF-1 (thyroid transcription factor-1) is a gene thatplays an active role in the lung development, and regulates thesurfactant protein-C gene expression. Protein TAZ is considered to be atranscription coactivator that facilitates the surfactant protein-C geneexpression by binding with TTF-1 [Park et al., J. Biol. Chem., 2004;279:17384-90]. Further, protein TAZ is suggested to regulate theexpression of TEF-1 regulatory genes in muscle tissues by binding withTEF-1 (transcription enhancer factor-1) [Mahoney et al., Biochem. J.,2005; 388:217-25].

Aside from the TAZ function of modulating mesenchymal stem celldifferentiation, other functions thereof in the migration, invasion, andtumorigenesis of breast cancer cells MCF7 are known [Chan et al., CancerRes., 2008; 68:2592-8]. The occurrence of polycystic kidney diseasewhich forms multiple renal cysts has been reported in TAZ-deficientanimal models, and various approaches for the functions of protein TAZhave been attempted [Makita et al., Am. J. Physiol. Renal. Physiol.,2008; 294:F542-53; Tian et al., Molecular & Cellular Biology, 2007;27:6383-95]. Meanwhile, there have been some reports that FGF-2 causesthe decrease of the TAZ protein in osteogenic differentiation [Deng Z Let al., Front Biosci., 2008; 13:2001-21; Eda et al., Biochemical &Biophysical Research Communications, 2008; 366:471-5], but fewinvestigations on the regulatory mechanism of the TAZ protein have beenconducted despite the importance of functions of the TAZ protein.

In case of the TAZ protein which acts as a transcription co-activator toregulate transcription factors with DNA binding activity, the migrationthereof from cytosol to nucleus is prerequisite. Until now, it is knownthat the TAZ protein can migrate into the nucleus by serinedephosphorylation, and the inhibition of binding between proteins TAZand 14-3-3 is suggested as a method for increasing the migration of theTAZ protein into the nucleus. Thus, compounds which facilitate themigration of TAZ into the nucleus may be considered to have aninhibitory effect on the adipocyte differentiation and facilitates theosteoblast differentiation.

The present inventors have endeavored to seek out a compound effectivefor preventing or treating osteoporosis, diabetes, or hyperlipidemia,which has led the finding that phenyltetrazole derivatives are effectivein the treatment or prevention of osteoporosis by regulating the TAZprotein and also in the treatment of the obesity by inhibiting adipocytedifferentiation.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide apharmaceutical composition for preventing or treating osteoporosis,obesity, diabetes, or hyperlipidemia, comprising a phenyltetrazolederivative or a pharmaceutical acceptable salt thereof as an activeingredient.

In accordance with one aspect of the present invention, there isprovided a pharmaceutical composition for preventing or treatingosteoporosis, obesity, diabetes, or hyperlipidemia, comprising acompound of formula (I) or a pharmaceutical acceptable salt thereof asan active ingredient:

wherein, A is ethyl or n-butyl; R¹ is methyl, —CH₂OH, —CO₂CH₃, —CH₂F,—CH(OCH₃)₂, —CH₂OC(═O)CH₃, styryl or —CH₂OCH₂SCH₃; R² is H, Br, —CO₂CH₃,phenyl, pyridin-2-yl, pyridin-3-yl,

or styryl; R³ is H or methyl; X is CH or N; and P is H or—CH(CH₃)OCH₂CH₃.

The inventive pharmaceutical composition comprising phenyltetrazolederivatives of formula (I) is effective in preventing or treatingosteoporosis, obesity, diabetes, or hyperlipidemia, by regulatingprotein TAZ.

DETAILED DESCRIPTION OF THE INVENTION

Hereinafter, the pharmaceutical composition according to the presentinvention is described in detail.

The compound of formula (I) is preferably selected from the groupconsisting of:

-   {2-ethyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol,    represented by formula (II):

-   2-butyl-5-methyl-6-pyridin-3-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine,    represented by formula (III):

-   methyl    2-butyl-6-pyridin-2-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine-5-carboxylate,    represented by formula (IV):

-   {2-butyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol,    represented by formula (V):

-   2-butyl-5-fluoromethyl-6-(1-oxypyridin-2-yl)-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine,    represented by formula (VI):

-   (2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-6-yl)phenylmethanol,    represented by formula (VII):

-   {2-butyl-5-dimethoxymethyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-6-yl}phenylmethanol,    represented by formula (VIII):

-   6-bromo-2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-5-ylmethyl    acetate, represented by formula (IX):

-   6-bromo-2-butyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-ylmethyl    acetate, represented by formula (X):

-   2-butyl-7-methyl-5-((methylsulfanylmethoxy)methyl)-6-phenyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine,    represented by formula (XI):

-   methyl    2-butyl-6-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate,    represented by formula (XII):

-   methyl    2-butyl-5-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-6-carboxylate    represented by formula (XIII):

The compounds of formulas (II) to (XIII) are more explained as follows:

TABLE 1 Structures and characteristics of the compounds of the presentinvention Compound (formula) Structure References or ¹H-NMR data(ppm) II

U.S. Pat. No. 5,332,744; EP 400974; and WO 95/34564. III

WO 95/021838, WO 95/34564; KR Patent Publication No. 95-25039; U.S. Pat.No. 5,849,753; and Bioorg. Med. Chem., 7(12), 2971 (1999). IV

WO 95/021838; KR Patent Publication No. 95-25039; U.S. Pat. No.5,849,753; EP 0743943; Bioorg. Med. Chem., 7(12), 2971 (1999); and Chem.Med. Chem., 2(9), 1298-1310, (2007). V

U.S. Pat. No. 5,849,753; EP 0765328; WO 95/21838; and KR PatentPublication No. 95-25039. VI

¹H-NMR(300 MHz, CDCl₃) δ 0.78(t, 3H), 1.29(m, 2H), 1.60(br-s, 2H),2.66(br-s, 2H), 5.10 and 5.45(d, 2H), 5.60(br-s, 2H), 6.73(d, 2H),6.95(d, 2H), 7.15(d, 1H), 7.39- 7.48(m, 5H), 7.48(d, 1H), 7.75(s, 1H),8.38(d, 1H) VII

¹H-NMR(300 MHz, CDCl₃) δ 0.90(t, 3H), 1.07(t, 3H), 1.38(m, 2H). 1.65(d,3H), 1.77(m, 2H), 2.74(t, 2H), 3.22(m, 1H), 3.43(m, 1H), 3.49(s, 3H),3.59(s, 3H), 3.67(s, 1H), 5.47(s, 2H), 5.52(s, 1H), 5.88(q, 1H), 6.77(s,1H), 7.11(m, 4H), 7.39(m, 3H), 7.50(m, 4H), 7.70(s, 1H), 7.87(d, 1H), J= 7.2 Hz) VIII

¹H-NMR(300 MHz, CDCl₃) δ 0.85(t, 3H), 1.31(m, 2H), 1.65(m, 2H), 2.63(t,2H), 3.41(s, 3H), 3.50(s, 3H), 5.38(m, 2H), 5.54(s, 1H), 6.64(s, 1H),6.97(m, 4H), 7.36(m, 3H), 7.41(d, 2H, J = 7.1 Hz), 7.53(m, 2H), 7.65(s,1H), 7.93(d, 1H, J = 7.5 Hz) IX

¹H-NMR(300 MHz, CDCl₃) δ 8.16(s, 1H), 7.88(dd, J = 7.5, 1.6 Hz, 1H),7.50(m, 2H), 7.41(dd, J = 7.5, 1.6 Hz, 1H), 7.14(d, J = 8.4 Hz, 2H),7.08(d, J = 8.4 Hz, 2H), 5.88(q, 1H), 5.42(m, 4H), 3.42(m, 1H), 3.20(m,1H), 2.80(t, 2H), 2.14(s, 3H), 1.78(m 2H), 1.63(d, J = 6.0 Hz, 3H),1.41(m, 2H), 1.05(t, 3H), 0.92(t, 3H). X

¹H-NMR(300 MHz, CDCl₃) δ 7.98(d, J = 7.3 Hz, 1H), 7.58-7.67(m, 3H),7.42(d, J = 7.3 Hz, 1H), 7.03(d, J = 6.8 Hz, 2H), 6.94(d, J = 6.8 Hz,2H), 5.41(s, 2H), 5.36(s, 2H), 2.73(t, 2H), 2.10(s, 3H), 1.69(m, 2H),1.35(m, 2H), 0.89(t, 2H). XI

¹H NMR(300 MHz, CDCl₃) δ 0.95(t, 3H), 1.43(m, 2H), 1.80(m, 2H), 2.07(s,3H), 2.35(s, 3H), 2.85(t, 2H), 4.55(s, 2H), 4.85(s, 2H), 5.30(s, 2H),7.00(m, 4H), 7.20(m, 2H), 7.32(m, 2H), 7.45(m, 5H) XII

¹H-NMR(300 MHz, CDCl₃) δ 0.83(t, 3H), 1.29(m, 2H), 1.59(m, 2H), 2.41(t,2H), 3.89(s, 3H), 5.29(s, 2H), 6.71(d, 2H, J = 8.1 Hz), 6.76(d, 1H, J =16.1 Hz), 6.91(d, 2H, J = 8.1 Hz), 7.23-7.32(m, 5H), 7.37(s, 1H),7.47(s, 1H), 7.49(m, 1H), 7.54-7.65(m, 2H), 7.90(d, 1H, J = 16.1 Hz),8.00(dd, 1H) XIII

¹H-NMR(300 MHz, CDCl₃) δ 0.86(t, 3H), 1.30(m, 2H), 1.62(m, 2H), 2.44(t,2H), 3.88(s, 3H), 5.26(s, 2H), 6.62(d, 1H, J = 16.0 Hz), 6.71(d, 2H, J =8.1 Hz), 6.92(d, 2H, J = 8.1 Hz), 7.14(s, 1H), 7.29-7.34(m, 2H),7.37-7.43(m, 2H), 7.49-7.55(m, 4H), 7.62(s, 1H), 7.85(d, 1H, J = 16.0Hz), 7.98(m, 1H)

The compounds of formulas (II) to (V) can be prepared by the methodsaccording to the conventional methods. Hereinafter, the preparationmethods of the compounds of formulas (VI) to (XIII) are described indetail.

The compound of formula (VI) may be prepared according to ReactionScheme 1:

<Step 1>

The compound of formula (I-1) is subjected to an alkylation reactionwith the compound of formula (I-2) in a solvent under a base conditionto obtain the compound of formula (I-3). Preferably, the base is sodiumhydroxide, sodium carbonate, potassium carbonate, or triethylamine, andthe solvent is dimethylformamide (DMF), acetonitrile, or tetrahydrofuran(THF). The reaction may be conducted for 3 hours at room temperature.

<Step 2>

The compound of formula (I-3) is dissolved in dichloromethane and DAST(diethylaminosulfur trifluoride, 1.2 eq) is added dropwise thereto at−78° C. The resulting solution is then slowly warmed to 0° C. andallowed to react for 10 minutes to obtain the compound of formula (I-4).

<Step 3>

The compound of formula (I-4) is dissolved in toluene, and 2-tributyltinpyridine (1-3 eq) and tetrakis(triphenylphosiphine) palladium (0.03 eq)are added thereto. The resulting solution is kept at 120° C. for 15hours to obtain the compound of formula (I-5).

<Step 4>

The compound of formula (I-5) is dissolved in a solvent, an oxidizingagent (1.3-5 eq) is added thereto, and the resulting solution is kept atroom temperature for 1 to 10 hours to obtain the compound of formula(I-6). Preferably, the oxidizing agent is m-chloroperoxybenzoic acid(MCPBA), oxone, hydroperoxide, or peracetic acid, and the solvent isdichloromethane, acetone, methanol, acetic acid, or water. The reactionis preferably conducted in solvents of MCPBA (1.5 eq) anddichloromethane at room temperature for 5 hours.

<Step 5>

The compound of formula (I-6) is dissolved in methanol, ethanol, ortetrahydrofuran, an acid (1-5 eq) is added thereto, and the resultingsolution is kept at room temperature for 10 minutes to 3 hours to obtainthe compound of formula (VI) whose tetrazolyl group is unprotected.Preferably, the acid is anhydrous hydrochloric acid, hydrochloride,p-toluenesulfonic acid, MeSO₃H, or acetic acid.

The compounds of formulas (I-1) and (I-2) may be prepared according tothe methods disclosed in WO 95/21838, WO 95/34564, and KR PatentLaid-open publications Nos. 96-00884 and 95-25039.

The compounds of formulas (VII) and (VIII) may be prepared according toReaction scheme 2:

<Step 1>

The procedure of Step 1 of Reaction Scheme 1 is repeated except forusing the compound of formula (II-1) as a starting material to obtainthe compound of formula (II-2).

<Step 2>

The compoound of formula (II-2) is dissolved in N,N-dimethylformamide,1-phenylvinylboronic acid (1-1.5 eq) is added thereto, and thenPd(PPh₃)₄ (5 mol %), 3M Na₂CO₃ (2 eq) or Pd(OAc)₂ (3 mol %), PPh₃ (10mol %) and triethylamine (2 eq) are added thereto. The resulting mixtureis kept at 110° C. for 5 hours to obtain the compound of formula (II-3).

<Step 3>

The compound of formula (II-3) is dissolved in 1,4-dioxane/water (3:1),and then OsO₄ (3-10 mol %) and NaIO₄ (2-3 eq) are added thereto. Theresulting mixture is kept at room temperature for 3 hours to obtain thecompound of formula (II-4). Otherwise, the resulting mixture isdissolved in dichloromethane and then kept at −78° C. for 2 hours withadding ozone gas to obtain the compound of formula (II-4).

<Step 4>

The compound of formula (II-4) is dissolved in tetrahydrofuran ordiethyl ether, and then PhMgBr or PhMgCl (1-2 eq) is added thereto. Theresulting mixture is kept at a temperature range of −78° C. to 0° C. foran hour to obtain the compound of formula (VII).

<Step 5>

The procedure of Step 5 of Reaction Scheme 1 is repeated except forusing the compound of formula (VII) as a starting material to obtain thecompound of formula (VIII).

The compounds of formulas (II-1) may be prepared according to themethods disclosed in WO 95/21838 and KR Patent Laid-open publicationsNo. 95-25039.

The compounds of formulas (IX) and (X) may be prepared according toReaction scheme 3:

<Step 1>

The procedure of Step 1 of Reaction Scheme 1 is repeated except forusing the compound of formula (III-1) as a starting material to obtainthe compound of formula (IX).

<Step 2>

The procedure of Step 5 of Reaction Scheme 1 is repeated except forusing the compound of formula (IX) as a starting material to obtain thecompound of formula (X).

The compounds of formulas (III-1) may be prepared according to themethods disclosed in WO 95/21838, U.S. Pat. No. 5,691,348, and KR PatentLaid-open publications No. 95-25039.

The compound of formula (XI) may be prepared according to Reactionscheme 4:

<Step 1>

The procedure of Step 1 of Reaction Scheme 1 is repeated except forusing the compound of formula (IV-1) as a starting material to obtainthe compound of formula (IV-2).

<Step 2>

The compound of formula (IV-2) is dissolved in N,N-dimethylformamide,and NaH (1.5 eq) is added thereto as a base, and then ClCH₂SMe (1.1 eq)and sodium iodide (0.3-1 eq) are added thereto. The resulting mixture iskept at a temperature range of 0° C. to room temperature for 3 hours toobtain the compound of formula (IV-3).

<Step 3>

The procedure of Step 5 of Reaction Scheme 1 is repeated except forusing the compound of formula (IV-3) as a starting material to obtainthe compound of formula (XI).

The compounds of formulas (IV-1) may be prepared according to themethods disclosed in WO 95/21838, U.S. Pat. No. 5,691,348, and KR PatentLaid-open publications No. 95-25039.

The compounds of formulas (XII) and (XIII) may be prepared according toReaction scheme 5:

wherein, R¹ and R² are each independently Br or COOMe (in proviso thatR¹ and R² are not same), and where R¹ in formula (V-2) is Br, R¹ informula (V-3) is styryl, and where R¹ in formula (V-2) is COOMe, R² informula (V-3) is styryl.

<Step 1>

The procedure of Step 1 of Reaction Scheme 1 is repeated except forusing the compound of formula (V-1) as a starting material to obtain thecompound of formula (V-2). The isomer may be obtained in the sameamount.

<Step 2>

The procedure of Step 2 of Reaction Scheme 2 is repeated except forusing the compound of formula (V-2) as a starting material to obtain theSuzuki coupled compound of formula (V-3).

<Step 3>

The procedure of Step 5 of Reaction Scheme 1 is repeated except forusing the compound of formula (V-3) as a starting material to obtain thecompounds of formulas (XII) and (XIII).

The compounds of formulas (V-1) may be prepared according to the methodsdisclosed in WO 95/21838, U.S. Pat. No. 5,691,348, and KR PatentLaid-open publication No. 95-25039.

The compound of formula (I) may be used in the form of apharmaceutically acceptable addition salt formed with a free acid suchas an organic or inorganic acid. Examples of such inorganic acid includehydrochloric acid, bromic acid, sulfuric acid, sulfurous, and phosphoricacids, preferably hydrochloric acid, while the organic acid may becitric acid, acetic acid, maleic acid, fumaric acid, gluconic acid,methanesulfonic acid, glycolic acid, succinic acid, tartaric acid,4-toluenesulfonic acid, galacturonic acid, embonic acid, glutamic acid,and aspartic acids, preferably methanesulfonic acid.

The addition salt according to the present invention may be prepared bya conventional method, e.g., by dissolving the compound of formula (I)in a water-miscible organic solvent (e.g., acetone, methanol, ethanol,and acetonitrile) and adding thereto an organic or inorganic acidspecified above in an equivalent or excessive amount, followed by theprecipitation or crystallization, or evaporating a solvent or an excessacid, followed by the drying or the filtration of precipitated salts ata reduced pressure.

It should be understood to those skilled in the art that the presentinvention comprises not only the compound of formula (I) and apharmaceutically acceptable salt thereof, but also a solvate, a hydrate,and a stereoisomer capable of being prepared therefrom within the scopeof the present invention.

The compound of formula (I) of the present invention is characterized bythe use for the prevention or treatment of osteoporosis, obesity,diabetes, or hyperlipidemia. The compounds of the present inventionfacilitate the translocation of transcription factor co-activator,protein TAZ into the nucleus (see Test 1), and the TAZ proteintranslocated not only inhibit the activity of PPARγ by binding eachother to inhibit adipocyte differentiation (see Test 2), but alsofacilitate the RUNX2 activity by binding each other to facilitateosteoblast differentiation (see Test 3). Further, in experiments invitro, the compounds of the present invention show the inhibition ofadipocyte differentiation and the facilitation of osteoblastdifferentiation (see Test 4). Thus, the compound of formula (I) of thepresent invention can be used in the prevention or treatment ofosteoporosis, obesity, diabetes, or hyperlipidemia.

The pharmaceutical compositions of the invention may be formulated foradministration orally or parenterally.

The composition for oral administration may take various forms such astablets, pill, soft and hard gelatin capsules, aqueous solutions,suspensions, emulsions, syrups, granules and elixirs, which may containconventional additives such as a diluent (e.g., lactose, dextrose,sucrose, mannitol, sorbitol, cellulose and glycine), a lubricant (e.g.,silica, talc, stearic acid or its magnesium or calcium salt, andpolyethylene glycol). The tablet may also contain a binder (e.g.,magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethyl cellulose, and polyvinyl pyrrolidone)and optionally a disintegrant (e.g., starch, agar, and alginic acid orits sodium salt), absorbent, colorant, flavor, sweetener and the like.

The pharmaceutical compositions of the invention may be formulated foradministration orally or parenterally, including subcutaneous,intravenous, intraperitoneal or intrathoracic injection. The parenteralformulation may be prepared in the unit dosage form by mixing thecompound of formula (I) or a pharmaceutically acceptable salt with astabilizer or buffer in water to obtain a solution or suspension, andpackaging into ampler or vials.

The composition may be sterilized and/or contain an adjuvant such as apreservative, stabilizer, wetting agent, emulsifier, a salt forcontrolling an osmotic pressure and/or a buffer solution, and otherpharmaceutically effective materials, and may be formulated by any ofconventional methods such as mixing, granulation, or coating.

The following Examples illustrate the present invention in more detail.However, these are merely examples, and the present invention is notlimited thereto.

The molecular structures of the present invention were confirmed byinfrared spectrometry, NMR spectroscopy, mass spectroscopy, liquidchromatography, X-ray crystallography, or comparison of ultimateanalysis values for a representative compound with a real measurementvalues.

Example 1 Preparation of2-butyl-5-fluoromethyl-6-(1-oxypyridin-2-yl)-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine(formula VI) <1-1> Preparation of(2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-6-bromo-3H-imidazo[4,5-b]pyridine-5-yl)methyanol(formula I-3)

(2-butyl-6-bromo-3H-imidazo[4,5-b]pyridine-5-yl)methanol (0.70 g, 2.49mmol) was dissolved in 10 mL of N,N-dimethylformamide, and thenpotassium carbonate (1.03 g. 7.5 mmol) and5-(4′-(bromomethyl)biphenyl-2-yl)-1-(1-ethoxyethyl)-1H-tetrazole (1.2 g,3.0 mmol) were added to the resulting solution, followed by stirring themixture at room temperature for 5 hours. 60 mL of water was added to thereaction mixture to dilute and extracted with ethyl acetate (60 mL×2).The organic layer was dried over anhydrous sodium sulfate and filtered,and then the solvent was removed. The resulting residue was concentratedunder reduced pressure and refined by silica gel column chromatography(n-hexane:ethyl acetate=2:1) to obtain the title compound (0.92 g,yield: 63%.)

¹H-NMR (300 MHz, CDCl₃) δ 0.94 (t, 3H), 1.06 (t, 3H), 1.43 (m, 2H), 1.64(d, 3H), 1.82 (m, 2H), 2.84 (t, 2H), 3.23 (m, 1H), 3.42 (m, 1H), 4.18(t, 1H, OH), 4.71 (d, 2H), 5.52 (s, 2H), 5.87 (q, 1H), 7.09-7.17 (m,4H), 7.32-7.53 (m, 3H), 7.86 (d, 1H), 7.89 (s, 1H); MS (m/e, M): 590.

<1-2> Preparation of methanesulfonic acid2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-6-bromo-5-fluoromethyl-3H-imidazo[4,5-b]pyridine(formula I-4)

The compound of formula (I-3) (0.56 g, 0.95 mmol) obtained in step <1-1>was dissolved in 10 mL of dichloromethane, and then cooled to −78° C.and diethylaminosulfur trifluoride (DAST, 0.14 mL, 1.05 mmol) was slowlyadded dropwise thereto. The resulting solution was warmed to 0° C.,stirred for about another 10 minutes and water was added thereto tofinish the reaction. The reaction mixture was diluted with 50 mL ofwater and extracted with ethyl acetate (50 mL×2). The organic layer wasdried over anhydrous sodium sulfate and filtered, and then the solventwas removed. The resulting residue was concentrated under reducedpressure and refined by silica gel column chromatography (n-hexane:ethylacetate=2:1) to obtain the title compound (0.32 g, yield: 57%.)

¹H-NMR (300 MHz, CDCl₃) δ 0.90 (t, 3H), 1.06 (t, 3H), 1.39 (m, 2H), 1.63(d, 3H), 1.76 (m, 2H), 2.80 (t, 2H), 3.20 (m, 1H), 3.42 (m, 1H), 5.46(s, 2H), 5.59 and 5.75 (s, 1H, CH2F), 5.87 (q, 1H), 7.05 (d, 2H), 7.13(d, 2H), 7.37-7.53 (m, 3H), 7.86 (dd, 1H), 8.18 (s, 1H); MS (m/e, M⁺):592.

<1-3> Preparation of2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-5-fluoromethyl-6-pyridine-2-yl-3H-imidazo[4,5-b]pyridine(formula I-5)

The compound of formula (I-4) (200 mg, 0.34 mmol) obtained in step <1-2>was dissolved in 10 mL of toluene, and then 2-tributyltin pyridine (250mg, 0.68 mmol) and Pd(PPh₃)₄ (20 mg, 0.017 mmol) were added thereto,followed by reacting at 120° C. for 16 hours. The solvent was removedand the resulting residue was concentrated under reduced pressure andthen refined by silica gel column chromatography (1. n-hexane:ethylacetate (1:1); 2. ethyl acetate) to obtain the title compound as an oil(160 mg, yield: 80%).

¹H-NMR (300 MHz, CDCl₃) δ 0.92 (t, 3H), 1.07 (t, 3H), 1.41 (m, 2H), 1.64(d, 3H), 1.81 (m, 2H), 2.83 (t, 2H), 3.20 (m, 1H), 3.42 (m, 1H), 5.54(dd, 2H), 5.58 and 5.74 (s, 1H, CH2F), 5.87 (q, 1H), 7.12 (d, 2H), 7.15(d, 2H), 7.41-7.61 (m, 5H), 7.84 (m, 2H), 8.13 (s, 1H), 8.72 (d, 1H); MS(m/e, M⁺): 590.

<1-4> Preparation of2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-5-fluoromethyl]-6-(1-oxypyridin-2-yl)-3H-imidazo[4,5-b]pyridine(formula I-5)

The compound of formula (I-5) (240 mg, 0.40 mmol) obtained in step <1-3>was dissolved in 5 mL of dichloromethane, and then m-chloroperoxybenzoicacid (m-CPBA, 200 mg, 0.81 mmol) was added thereto, followed by stirringat room temperature for 3 hours. The solvent was removed and theresulting residue was concentrated under reduced pressure and thenrefined by silica gel column chromatography (1. n-hexane:ethyl acetate(1:1); 2.5% methanol/dichloromethane) to obtain the title compound as asolid (170 mg, yield: 70%).

¹H-NMR (300 MHz, CDCl₃) d 0.92 (t, 3H), 1.08 (t, 3H), 1.42 (m, 2H), 1.65(d, 3H), 1.80 (m, 2H), 2.83 (t, 2H), 3.20 (m, 1H), 3.42 (m, 1H), 5.52(s, 2H), 5.46 and 5.62 (s, 1H, CH₂F), 5.87 (q, 1H), 7.13-7.14 (m, 4H),7.37-7.53 (m, 6H), 7.86 (dd, 1H), 8.00 (s, 1H), 8.45 (d, 1H); MS (m/e,M⁺): 606.

<1-5> Preparation of2-butyl-5-fluoromethyl-6-(1-oxypyridin-2-yl)-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine(formula VI)

The compound of formula I-6 (120 mg, 0.19 mmol) obtained in step <1-4>was dissolved in 3 mL of methanol, and then 1 mL of 3N—HCl was addedthereto, followed by stirring at room temperature for 20 minutes. Thereaction mixture is adjust to about pH 4 by adding 1N—NaOH, diluted in20 mL of water and extracted with ethyl acetate (20 mL×2), and then theorganic layer was dried over anhydrous sodium sulfate and filtered. Thesolvent was removed, and the resulting residue was concentrated underreduced pressure and then refined by n-hexane/ethyl acetate to obtainthe title compound (100 mg, yield: 94%).

¹H-NMR (300 MHz, CDCl₃) δ 0.78 (t, 3H), 1.29 (m, 2H), 1.60 (br-s, 2H),2.66 (br-s, 2H), 5.10 and 5.45 (d, 2H), 5.60 (br-s, 2H), 6.73 (d, 2H),6.95 (d, 2H), 7.15 (d, 1H), 7.39-7.48 (m, 5H), 7.48 (d, 1H), 7.75 (s,1H), 8.38 (d, 1H); FAB-MS (m/e, M⁺): 535 (M⁺+1).

Example 2 Preparation of(2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-6-yl)phenylmethanol(formula VII) <2-1> Preparation of6-bromo-2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridine(formula II-2)

The procedure of <1-1> was repeated except for using6-bromo-2-butyl-5-dimethoxymethyl-3H-imidazo[4,5-b]pyridine (1.8 g, 5.48mmol) instead of a compound of formula (I-1) to obtain the titlecompound (1.91 g, yield: 55%).

¹H-NMR (300 MHz, CDCl₃) δ 0.92 (t, 3H), 1.06 (t, 3H), 1.42 (m, 2H), 1.63(d, 3H), 1.79 (m, 2H), 2.79 (t, 2H), 3.21 (m, 1H), 3.42 (m, 1H), 3.48(s, 6H), 5.47 (s, 2H), 5.79 (s, 1H), 5.86 (q, 1H), 7.08 (d, 2H), 7.12(d, 2H), 7.37 (dd, 1H), 7.44-7.55 (m, 2H), 7.85 (dd, 1H), 8.16 (s, 1H);MS (m/e, M⁺): 634.

<2-2> Preparation of2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-6-styryl-3H-imidazo[4,5-b]pyridine(formula II-3)

The compound of formula (II-2) obtained in <2-1> (0.5 g, 0.79 mmol) wassuspended in 10 mL of 1,2-dimethoxyethane, and 350 mg oftrans-2-phenylvinylboronic acid (2.37 mmol, 3 eq), 46 mg of Pd(PPh₃)₄(0.04 mmol, 0.05 eq) and 0.79 mL of 3M-Na₂CO₃ (2.37 mmol, 3 eq) wereadded thereto, followed by stirring with reflux at 90° C. for 5 hours.The resulting mixture was diluted with 60 mL of ethyl acetate, filteredthrough celite, and concentrated by evaporation under a reducedpressure. The resulting residue was purified by silica gel columnchromatography (n-hexane:ethyl acetate=2:1) to obtain the title compoundas a yellowish solid (355 mg, yield: 68%).

¹H-NMR (300 MHz, CDCl₃): δ 0.93 (t, 3H), 1.08 (t, 3H), 1.43 (m, 2H),1.66 (d, 3H), 1.81 (m, 2H), 2.80 (t, 2H), 2.80 (t, 2H), 3.23 (m, 1H),3.46 (m, 1H), 3.48 (s, 6H), 5.49 (d, 2H), 5.55 (s, 1H), 5.87 (q, 1H),7.00 (d, 1H, J=16.2 Hz), 7.11 (m, 3H), 7.29 (m, 2H), 7.39 (m, 3H), 7.51(m, 2H), 7.55 (m, 2H), 7.88 (m, 2H), 8.29 (s, 1H); MS (m/e, M⁺): 657.

<2-3> Preparation of2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridine-6-carbaldehyde(formula II-4)

The compound of formula (II-3) obtained in <2-2> (355 mg, 0.54 mmol) wassuspended in 6 mL of 1,4-dioxane and 2 mL of water, and 346 mg of NaIO₄(1.62 mmol, 3 eq.) and OsO₄ (2.5 wt % in 2-methyl-2-propanol) ascatalysts were added thereto, followed by stirring at room temperaturefor 2 hours. Then, the resulting mixture was diluted with 15 mL of waterand washed with water and a brine while extracting with ethyl acetate(15 mL). The extracted organic layer was dried over anhydrous sodiumsulfate, filtered, and concentrated by evaporation under a reducedpressure. The resulting residue was purified by silica gel columnchromatography (n-hexane:ethyl acetate=1:1) to obtain the title compoundas a yellowish oil (255 mg, yield: 81%).

¹H-NMR (300 MHz, CDCl₃): δ 0.92 (t, 3H), 1.07 (t, 3H), 1.41 (m, 2H).1.65 (d, 3H), 1.80 (m, 2H), 2.81 (t, 2H), 3.23 (m, 1H), 3.45 (m, 1H),3.51 (s, 6H), 5.51 (s, 1H), 5.89 (q, 1H), 7.08 (d, 2H, J=8.1 Hz), 7.14(d, 2H, J=8.3 Hz), 7.40 (dd 1H, J=1.7, 7.5 Hz), 7.51 (m, 2H), 7.87 (dd,1H, J=1.7, 7.5 Hz), 8.61 (s, 1H), 10.75 (s, 1H, —CHO); MS (m/e, M⁺):583.

<2-4> Preparation of(2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridine-6-yl)phenyl-methanol(formula VII)

The compound of formula (II-4) obtained in <2-3> (255 mg, 0.44 mmol) wassuspended in 6 mL of tetrahydrofuran, and 0.44 mL of phenyl magnesiumbromide solution (3.0M solution dissolved in diethyl ether) (1.31 mmol,3 eq.) was added thereto at −78° C., followed by stirring at sametemperature for 30 min. Then, the resulting mixture was diluted with 15mL of water and washed with water and a brine while extracting withethyl acetate (15 mL). The extracted organic layer was dried overanhydrous sodium sulfate, filtered, and concentrated by evaporationunder a reduced pressure. The resulting residue was purified by silicagel column chromatography (n-hexane:ethyl acetate=1:2) to obtain thetitle compound as whitish foam (220 mg, yield: 76%).

¹H-NMR (300 MHz, CDCl₃): δ 0.90 (t, 3H), 1.07 (t, 3H), 1.38 (m, 2H).1.65 (d, 3H), 1.77 (m, 2H), 2.74 (t, 2H), 3.22 (m, 1H), 3.43 (m, 1H),3.49 (s, 3H), 3.59 (s, 3H), 3.67 (s, 1H), 5.47 (s, 2H), 5.52 (s, 1H),5.88 (q, 1H), 6.77 (s, 1H), 7.11 (m, 4H), 7.39 (m, 3H), 7.50 (m 4H),7.70 (s, 1H), 7.87 (d, 1H, J=7.2 Hz); MS (m/e, M⁺): 661.

Example 3 Preparation of{2-butyl-5-dimethoxymethyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-6-yl}phenylmethanol(formula VIII)

The procedure of <1-5> was repeated except for using the compound offormula (VII) (100 mg, 0.15 mmol) instead of a compound of formula (I-6)to obtain the title compound (80 mg, yield: 90%).

¹H-NMR (300 MHz, CDCl₃): δ 0.85 (t, 3H), 1.31 (m, 2H), 1.65 (m, 2H),2.63 (t, 2H), 3.41 (s, 3H), 3.50 (s, 3H), 5.38 (m, 2H), 5.54 (s, 1H),6.64 (s, 1H), 6.97 (m, 4H), 7.36 (m, 3H), 7.41 (d, 2H, J=7.1 Hz), 7.53(m, 2H), 7.65 (s, 1H), 7.93 (d, 1H, J=7.5 Hz); MS (m/e, M⁺): 589.

Example 4 Preparation of6-bromo-2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate (formula IX)

The procedure of <1-1> was repeated except for using6-bromo-2-butyl-3H-imidazo[4,5-b]pyridine-5-ylmethyl acetate of formula(III-1) (300 mg, 0.92 mmol) instead of a compound of formula (I-1) toobtain the title compound (340 mg, yield: 59%).

¹H-NMR (300 MHz, CDCl₃): δ 8.16 (s, 1H), 7.88 (dd, J=7.5, 1.6 Hz, 1H),7.50 (m, 2H), 7.41 (dd, J=7.5, 1.6 Hz, 1H), 7.14 (d, J=8.4 Hz, 2H), 7.08(d, J=8.4 Hz, 2H), 5.88 (q, 1H), 5.42 (m, 4H), 3.42 (m, 1H), 3.20 (m,1H), 2.80 (t, 2H), 2.14 (s, 3H), 1.78 (m 2H), 1.63 (d, J=6.0 Hz, 3H),1.41 (m, 2H), 1.05 (t, 3H), 0.92 (t, 3H).

Example 5 Preparation of6-bromo-2-butyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate (formula X)

The procedure of <1-5> was repeated except for using the compound offormula (IX) obtained in Example 4 (150 mg, 0.23 mmol) instead of acompound of formula (I-6) to obtain the title compound (126 mg, yield:95%).

¹H-NMR (300 MHz, CDCl₃) δ 7.98 (d, J=7.3 Hz, 1H), 7.58-7.67 (m, 3H),7.42 (d, J=7.3 Hz, 1H), 7.03 (d, J=6.8 Hz, 2H), 6.94 (d, J=6.8 Hz, 2H),5.41 (s, 2H), 5.36 (s, 2H), 2.73 (t, 2H), 2.10 (s, 3H), 1.69 (m, 2H),1.35 (m, 2H), 0.89 (t, 2H).

Example 6 Preparation of2-butyl-7-methyl-5-[(methylsulfanylmethoxy)methyl]-6-phenyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine(formula XI) <6-1> Preparation of(2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-7-methyl-6-phenyl-3H-imidazo[4,5-b]pyridine-5-yl)-methanolformula IV-2)

The procedure of <1-1> was repeated except for using(2-butyl-7-methyl-6-phenyl-3H-imidazo[4,5-b]pyridine-5-yl)methanol offormula (IV-1) (0.5 g, 1.69 mmol) instead of a compound of formula (I-1)to obtain the title compound (0.58 g, yield: 57%).

¹H-NMR (300 MHz, CDCl₃): δ 0.92 (t, 3H), 1.08 (t 3H), 1.40 (m, 2H), 1.66(d, 3H), 1.78 (m, 2H), 2.35 (s, 3H), 2.75 (t, 2H), 3.24 (m, 1H), 3.42(m, 1H), 4.78 (d, 2H, J=5.3 Hz), 5.28 (t, 1H, —OH), 5.48 (s, 2H), 5.87(q, 1H), 7.15 (m, 4H), 7.21 (d, 2H, J=7.8 Hz), 7.39-7.54 (m, 6H) 7.88(dd, 1H, J=1.0, 7.3 Hz); Mass: 601.

<6-2> Preparation of2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-7-methyl-5-methylsulfanylmethoxymethyl-6-phenyl-3H-imidazo[4,5-b]pyridine(formula IV-3)

The compound of formula (IV-2) obtained in <6-1> (0.53 g, 0.88 mmol) wassuspended in 5 mL of N,N-dimethylformamide, cooled to 0° C., and sodiumhydride (60%; 53 mg, 1.32 mmol), chloromethyl methylsulfide (0.12 mL,1.32 mmol) and NaI (0.13 g, 0.88 mmol) were added thereto, followed bystirring at room temperature for 3 hours. Then, the resulting mixturewas diluted with 50 mL of water and extracted with ethyl acetate (50mL×2). The organic layer was dried over anhydrous sodium sulfate,filtered, and concentrated by evaporation under a reduced pressure. Theresulting residue was purified by silica gel column chromatography(n-hexane:ethyl acetate=3:1) to obtain the title compound (0.49 g,yield: 85%).

¹H-NMR (300 MHz, CDCl₃): δ 0.93 (t, 3H), 1.10 (t 3H), 1.38 (m, 2H), 1.70(d, 3H), 1.76 (m, 2H), 2.07 (s, 3H), 2.35 (s, 3H), 2.76 (t, 2H), 3.23(m, 1H), 3.45 (m, 1H), 4.50 (s, 2H), 4.95 (s, 2H), 5.45 (s, 2H), 5.84(q, 1H), 7.10 (m, 4H), 7.20 (d, 2H), 7.25-7.50 (m, 6H) 7.90 (m, 1H);Mass: 661.

<6-3> Preparation of2-butyl-7-methyl-5-[(methylsulfanylmethoxy)methyl]-6-phenyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine(formula XI)

The procedure of <1-5> was repeated except for using the compound offormula (IV-3) obtained in Example <6-2> (0.22 g, 0.33 mmol) instead ofa compound of formula (I-6) to obtain the title compound (0.18 g, yield:91%).

¹H-NMR (300 MHz, CDCl₃): δ 0.95 (t, 3H), 1.43 (m, 2H), 1.80 (m, 2H),2.07 (s, 3H), 2.35 (s, 3H), 2.85 (t, 2H), 4.55 (s, 2H), 4.85 (s, 2H),5.30 (s, 2H), 7.00 (m, 4H), 7.20 (m, 2H), 7.32 (m, 2H) 7.45 (m, 5H);Mass: 589.

Example 7 Preparation of methyl2-butyl-6-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate(formula XII) <7-1> Preparation of methyl6-bromo-2-butyl-1-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate(formula V-2a; R¹=Br, R²=COOMe) and methyl5-bromo-2-butyl-1-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl]-1H-benzimidazol-6-carboxylate(formula V-2b; R¹=COOMe, R²=Br)

The procedure of <1-1> was repeated except for using methyl6-bromo-2-butyl-1H-benzimidazol-5-carboxylate of formula (V-1) (2.5 g,8.0 mmol) instead of a compound of formula (I-1) to obtain the titlecompounds of formula (V-2a) (1.87 g, yield: 38%) and formula (V-2b)(1.97 g, yield: 40%) as oils.

¹H-NMR (300 MHz, CDCl₃) V-2a: δ 0.93 (t, 3H), 1.05 (t, 3H), 1.42 (m,2H), 1.65 (d, 3H), 1.82 (m, 2H), 2.83 (t, 2H), 3.22 (m, 1H), 3.43 (m,1H), 3.93 (s, 3H), 5.34 (s, 2H), 5.86 (q, 1H), 6.93 (d, 1H, J=8.1 Hz),7.16 (d, 1H, J=8.1 Hz), 7.46 (d, 1H, J=7.5 Hz), 7.46-7.57 (m, 2H), 7.50(s, 1H), 7.89 (d, 1H, J=7.5 Hz), 8.24 (s, 1H); Mass: 619.

¹H-NMR (300 MHz, CDCl₃) V-2b: δ 0.93 (t, 3H), 1.04 (t, 3H), 1.43 (m,2H), 1.64 (d, 3H), 1.81 (m, 2H), 2.82 (t, 2H), 3.21 (m, 1H), 3.42 (m,1H), 3.91 (s, 3H), 5.34 (s, 2H), 5.86 (q, 1H), 6.93 (d, 1H, J=8.0 Hz),7.14 (d, 1H, J=8.0 Hz), 7.40 (d, 1H, J=7.6 Hz), 7.46-7.56 (m, 2H), 7.76(s, 1H), 7.88 (d, 1H, J=7.6 Hz), 8.02 (s, 1H); Mass: 619.

<7-2> Preparation of methyl2-butyl-1-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl]-6-styryl-1H-benzimidazol-5-carboxylate(formula V-3a; R¹=styryl, R²=COOMe) and methyl2-butyl-1-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-5-styryl-1H-benzimidazol-6-carboxylate(formula V-3b; R¹=COOMe, R²=styryl)

The procedure of <2-2> was repeated except for using the compound offormula (V-2) obtained in Example <7-2> (1.0 g, 1.62 mmol) instead of acompound of formula (II-2) to obtain the title compounds of formula(V-3a) (0.72 g, yield: 70%) and formula (V-3b) (0.69 g, yield: 67%) asoils.

¹H-NMR (300 MHz, CDCl₃) V-3a: δ 0.92 (t, 3H), 1.01 (t, 3H), 1.45 (m,2H), 1.61 (d, 3H), 1.84 (m, 2H), 2.84 (t, 2H), 3.20 (m, 1H), 3.36 (m,1H), 3.92 (s, 3H), 5.39 (s, 2H), 5.80 (q, 1H), 6.85 (d, 1H, J=16.2 Hz),6.98 (d, 1H, J=8.2 Hz), 7.16 (d, 1H, J=8.2 Hz), 7.24 (m, 1H), 7.31-7.36(m, 2H), 7.41 (dd, 1H), 7.47-7.55 (m, 4H), 7.48 (s, 1H), 7.88 (dd, 1H),8.08 (d, 1H, J=16.1 Hz), 8.38 (s, 1H); Mass: 642.

¹H-NMR (300 MHz, CDCl₃) V-3b: δ 0.94 (t, 3H), 1.06 (t, 3H), 1.45 (m,2H), 1.64 (d, 3H), 1.85 (m, 2H), 2.83 (t, 2H), 3.22 (m, 1H), 3.42 (m,1H), 3.91 (s, 3H), 5.36 (s, 2H), 5.87 (q, 1H), 6.95 (d, 1H, J=8.2 Hz),6.98 (d, 1H, J=15.8 Hz), 7.27 (m, 1H), 7.33-7.58 (m, 7H), 7.87 (d, 1H,J=7.6 Hz), 7.90 (s, 1H), 8.05 (s, 1H), 8.07 (d, 1H, J=15.8 Hz); Mass:642.

<7-3> Preparation of methyl2-butyl-6-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate(formula XII)

The procedure of <1-5> was repeated except for using the compound offormula (V-3a) obtained in Example <7-2> (0.21 g, 0.32 mmol) instead ofa compound of formula (I-6) to obtain the title compound (0.154 g,yield: 85%).

¹H-NMR (300 MHz, CDCl₃): δ 0.83 (t, 3H), 1.29 (m, 2H), 1.59 (m, 2H),2.41 (t, 2H), 3.89 (s, 3H), 5.29 (s, 2H), 6.71 (d, 2H, J=8.1 Hz), 6.76(d, 1H, J=16.1 Hz), 6.91 (d, 2H, J=8.1 Hz), 7.23-7.32 (m, 5H), 7.37 (s,1H), 7.47 (s, 1H), 7.49 (m, 1H), 7.54-7.65 (m, 2H), 7.90 (d, 1H, J=16.1Hz), 8.00 (dd, 1H); Mass: 568.

Example 8 Preparation of methyl2-butyl-5-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-6-carboxylate(formula XIII)

The procedure of <1-5> was repeated except for using the compound offormula (V-3b) obtained in Example <7-2> (0.25 g, 0.39 mmol) instead ofa compound of formula (I-6) to obtain the title compound (0.19 g, yield:87%).

¹H-NMR (300 MHz, CDCl₃): δ 0.86 (t, 3H), 1.30 (m, 2H), 1.62 (m, 2H),2.44 (t, 2H), 3.88 (s, 3H), 5.26 (s, 2H), 6.62 (d, 1H, J=16.0 Hz), 6.71(d, 2H, J=8.1 Hz), 6.92 (d, 2H, J=8.1 Hz), 7.14 (s, 1H), 7.29-7.34 (m,2H), 7.37-7.43 (m, 2H), 7.49-7.55 (m, 4H), 7.62 (s, 1H), 7.85 (d, 1H,J=16.0 Hz), 7.98 (m, 1H); Mass: 568.

The substituents of the compounds of the present invention weresummarized and shown in following Table 2.

TABLE 2 Substituents of the compounds of formula (I) Compound (formulaNo) R¹ R² R³ A X P II —CH₂OH H Me ethyl N H III Me pyridin-3-yl Hn-butyl N H IV —CO₂Me pyridin-2-yl H n-butyl N H V —CH₂OH H Me n-butyl NH VI —CH₂F

H n-butyl N H VII —CH(OMe)₂

H n-butyl N

VIII —CH(OMe)₂

H n-butyl N H IX —CH₂OAc Br H n-butyl N

X —CH₂OAc Br H n-butyl N H XI —CH₂OCH₂SMe phenyl H n-butyl N H XII

COOMe H n-butyl CH H XIII —CO₂Me

H n-butyl CH H

The compounds of formulas (II) to (XIII) were evaluated forpharmaceutical efficacy according to the following procedures.

Test 1. Effect on Stimulating TAZ Protein Migration Toward Nucleus

Vector pEGFP-TAZ in which GFP (green fluorescence protein) is linked toprotein TAZ was prepared, and nucleus specific histone protein (RFP-H2B)expression vector having introduced RFP (red fluorescence protein)therein was prepared. The vector pEGFP-TAZ was prepared by introducingfull length TAZ cDNA synthesized by PCR method into vector pEGFP(Invitrogen, Carlsbad, Calif., USA) and then cloning. The expressionvector RFP-H2B was prepared by introducing full length histone H2B cDNAinto expression vector RFP (Clontech Laboratories, Inc., Palo Alto,Calif., USA) and then cloning.

Cos7 cells (ATCC, Manassas, Va.) were distributed to each well of a96-well plate (5×10³ cells/well) and stabilized, and then vectorspEGFP-TAZ and RFP-H2B were introduced into the cells by employingEffectene™ (Qiagen). After 48 hours, the cells were treated with each ofthe inventive compounds to a concentration of 10 μM. After 30 minutes,the movement of green fluorescent color toward the cell nucleus wasobserved continually by employing BD Pathway™ high-content bioimager (BDbioscience). Using a quantification program (BD IPLab™ for Pathway andBD™ Image Data Explorer), GFP expression in the nucleus of RFP-expressedcells was quantified.

GFP expressions of protein TAZ in the nucleus were analyzed andcalculated in percentage term based on that of non-treated cells in acontrol group, which are shown in Table 3.

TABLE 3 Effects on stimulating migration of protein TAZ toward nucleusCompound No II III IV V VI VII IX XI XII Expression 130.30 156.83 135.25150.56 132.42 160.30 147.45 133.70 149.46 (%)

As shown in Table 3, the inventive compounds promoted the migration ofprotein TAZ toward nucleus by more than 130%. Accordingly, the inventivecompounds are each expected to affect the differentiation intoadipocytes or osteoblasts

Test 2. Effect on TAZ Activity of Inhibiting PPARγ Function

293T cells (ATCC) were placed in the wells of a 48-well plate (1×10⁵cells/well) and stabilized. Introduced to the cells were PPARγ and TAZexpression vectors together with vectors aP2-luc (adipose fatty acidbinding protein 2 promoter linked to luciferase) and pCMVβ [Hong et al.,Science 2005; 309:1074-8]. For observing the effect on transcriptionactivation of the target gene by the PPARy or TAZ protein expression,aP2-luc reporter gene was also introduced into the cells. Forcalibration of the introduction efficiency in cells, vector pCMVβ wasintroduced in cells in same amounts and the β-galactosidase activity wasmeasured to calibrate the luciferase activity.

After 24 hours, the cells were treated with each of the inventivecompounds to a concentration of 10 μM and then further cultivated for 24hours. Proteins of the resulting cells were extracted with a lysissolution comprising NP-40, and thus luciferase activity was measured(Promega, Sunnyvale, Calif., USA).

Increased inhibitory effect on the aP2 promoter activity was analyzedand the results were calculated in percentage term based on theinhibitory effect on the aP2 promoter activity observed for thenon-treated cells in a control group. The results are shown in Table 4.

TABLE 4 Effects on TAZ activity inhibiting PPARγ function Compound No IIIII V VI VII VIII IX X XI XII XIII Increased 43.2 64.6 32.3 51.3 46.769.2 22.7 57.5 83.0 69.7 39.9 inhibition (%)

It is shown from Table 4 that the inventive compounds exhibitsignificant inhibitory effects, particularly the compounds of formulas(III), (VIII), (XI), and (XII) exhibit an inhibitory effect of more than60%. Accordingly, the inventive compounds inhibit PPARγ by binding toTAZ to inhibit the differentiation of PPARγ into adipocytes, and thusthey are useful for preventing or treating obesity.

Test 3. Effect on TAZ Activity of Stimulating RUNX2 Function

Introduced in 293T cells were introduced RUNX2 and TAZ expression vectortogether with 6xOSE-luc (osteocalcin-specific element linked toluciferase, six copies of RUNX2-binding site in the osteocalcin promoterlinked to luciferase) [Hong et al., Science 2005; 309:1074-8]. This testwas for an evaluation of an activity of the inventive compounds instimulating the luciferase activity which increases when RUNX2 binds toTAZ.

After 24 hours, the cells were treated with each of the inventivecompounds to a concentration of 10 μM and then further cultivated for 24hours. The procedure of Test 2 was repeated to extract the cell protein,and then reporter gene analysis was conducted to quantify the effect ofthe inventive compounds on the TAZ activity in stimulating RUNX2. Forcomparing the transformation efficaciency, β-galactosidase expressionvector (pCMVβ) was introduced to 293T cells and the β-galactosidaseactivity was measured for calibration.

The osteocalcin promoter activity was analyzed in percentage term basedon that of non-treated cells in a control group. When the value exceeds570% of the average increase by protein TAZ, the compound was consideredto have an additional stimulating activity. The results are shown inTable 5.

TABLE 5 Effects on TAZ activity of stimulating RUNX2 function CompoundNo II III IV V VI VII VIII IX X XI XII XIII Activity 472.6 259.4 463.2468.2 483.4 476.4 688.0 675.8 671.6 753.6 296.1 568.8 (%)

From Table 5, It can be seen that the additional stimulating activitywas observed for the inventive compounds, particularly the compounds offormulas (VIII), (IX), (X), and (XI). Accordingly, the inventivecompounds facilitate the RUNX2 activity by binding TAZ to stimulateosteoblast differentiation, and thus, they are useful for preventing ortreating osteoporosis.

Test 4. Effect on Differentiation into Adipocytes or Osteoblasts

The activity of the inventive compounds in promoting the differentiationof 3T3-L1 cells and C3H10T1/2 cells into adipocytes and osteoblasts, wasevaluated.

<4-1> Inducing Effect on Differentiation of 3T3-L1 Cells into Adipocytes

3T3-L1 preadipocytes (ATCC CL-173) were dispersed in a DMEM mediumcomprising 10% FBS (fatal bovine serum, and then confluently cultivatedfor 48 hours in a 24-well plate (3×10⁴ cells/well). 2 μM rosiglitazone,5 μg/mL insulin, and 1 μM dexamethasone were added thereto to induceadipocyte differentiation.

After 48 hours, the medium was replaced with a DMEM comprising 5 μg/mLinsulin and 10% FBS (fatal bovine serum), and 48 hours later, the mediumwas replaced with a DMEM comprising 10% FBS. Then, the medium wasreplaced every 48 hours to evaluate adipocyte differentiation. Theinventive compounds were each added during the replacement of themedium. After 8 days, the cells were fixed with 10% formalin, andOil-red O staining was conducted to confirm adipocytes generated incells.

The result of the staining showed that adipocyte differentiation wasinhibited by the inventive compounds, particularly the compounds offormulas (III), (IV), and (XII).

Further, in case of treating at a higher concentration, markedlyincreased inhibitory effect was observed for the compound of formula(VII).

<4-2> Inducing Effect on Differentiation of C3H10T1/2 Cells intoOsteoblasts

C3H10T1/2 cells (ATCC CCL 226) were diluted in a α-MEM medium comprising10% FBS, cultivated in a 96-well plate (1×10⁴ cells/well) for 48 hours,and the medium was replaced every 48 hours to observe the osteoblastdifferentiation. On the initiation of differentiation, the medium wastreated with 10 μM of each of the inventive compounds.

After 20 days, the cells were fixed with 70% ethanol and stained with anAlizarin red S solution to confirm increased calcium in osteocytes. Inthe Alizarin red staining test, the degrees of osteoblast differentioncan be identified by the intensity of red color.

The result of the staining showed that the osteoblast differentiationwas stimulated by the inventive compounds, particularly the compounds offormulas (III) and (V).

Accordingly, the inventive compounds are effective for inhibitingadipocyte differentiation and stimulating osteoblast differentiation,and thus they are useful for preventing or treating osteoporosis orobesity.

While the invention has been described with respect to the abovespecific embodiments, it should be recognized that various modificationsand changes may be made to the invention by those skilled in the artwhich also fall within the scope of the invention as defined by theappended claims.

1. A pharmaceutical composition for preventing or treating osteoporosis,comprising a compound of formula (I) or a pharmaceutical acceptable saltthereof as an active ingredient:

wherein, A is ethyl or n-butyl; R¹ is methyl, —CH₂OH, —CO₂CH₃, —CH₂F,—CH(OCH₃)₂, —CH₂C(═O)CH₃, styryl or —CH₂OCH₂SCH₃; R² is H, Br, —CO₂CH₃,phenyl, pyridin-2-yl, pyridin-3-yl,

 or styryl; R³ is H or methyl; X is CH or N; and P is H or—CH(CH₃)OCH₂CH₃.
 2. The pharmaceutical composition of claim 1, whereinthe compound of formula (I) is selected from the group consisting of:{2-ethyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol;2-butyl-5-methyl-6-pyridin-3-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;methyl2-butyl-6-pyridin-2-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine-5-carboxylate;{2-butyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol;2-butyl-5-fluoromethyl-6-(1-oxypyridin-2-yl)-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;(2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-6-yl)phenylmethanol;{2-butyl-5-dimethoxymethyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-6-yl}phenylmethanol;6-bromo-2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate;6-bromo-2-butyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate;2-butyl-7-methyl-5-((methylsulfanylmethoxy)methyl)-6-phenyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;methyl2-butyl-6-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate;and methyl2-butyl-5-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-6-carboxylate.3. A pharmaceutical composition for preventing or treating obesity,diabetes, or hyperlipidemia, comprising a compound of formula (I) or apharmaceutical acceptable salt thereof as an active ingredient:

wherein, A is ethyl or n-butyl; R¹ is methyl, —CH₂OH, —CO₂CH₃, —CH₂F,—CH(OCH₃)₂, —CH₂OC(═O)CH₃, styryl or —CH₂OCH₂SCH₃; R² is H, Br, —CO₂CH₃,phenyl, pyridin-2-yl, pyridin-3-yl,

 or styryl; R³ is H or methyl; X is CH or N; and P is H or—CH(CH₃)OCH₂CH₃.
 4. The pharmaceutical composition of claim 3, whereinthe compound of formula (I) is selected from the group consisting of:{2-ethyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol;2-butyl-5-methyl-6-pyridin-3-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;methyl2-butyl-6-pyridin-2-yl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine-5-carboxylate;{2-butyl-7-methyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-yl}methanol;2-butyl-5-fluoromethyl-6-(1-oxypyridin-2-yl)-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;(2-butyl-5-dimethoxymethyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-6-yl)phenylmethanol;{2-butyl-5-dimethoxymethyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-6-yl}phenylmethanol;6-bromo-2-butyl-3-{2′-[1-(1-ethoxyethyl)-1H-tetrazol-5-yl]biphenyl-4-ylmethyl}-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate;6-bromo-2-butyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridin-5-ylmethylacetate;2-butyl-7-methyl-5-((methylsulfanylmethoxy)methyl)-6-phenyl-3-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-3H-imidazo[4,5-b]pyridine;methyl2-butyl-6-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-5-carboxylate;and methyl2-butyl-5-styryl-1-[2′-(1H-tetrazol-5-yl)biphenyl-4-ylmethyl]-1H-benzimidazol-6-carboxylate.